Techniques for site-specific mutagenesis of cloned genes are well established; several variations are currently in use in many laboratories. Prior to the development of adequate recombinant DNA techniques, mutants could be obtained only by random mutagenesis requiring selection techniques specific for a particular phenotype. More modern techniques, however, provide scientists with other methods of altering DNA sequences by site-specific mutagenesis. Most of these techniques are summarized in Lathe, R. F. et al, Genetic Engineering, Academic Press, NY, Vol. 4, pp. 1-56 (1983). In essence, these techniques describe methods of manipulating DNA involving the transfer of DNA segments from one location to another. Such manipulation may permit the alteration of a DNA sequence in order to determine its function, or may permit the production of reagents with commercial or medical significance.
The present invention, an improvement on these techniques, is based on the discovery of three elements: (a) uracil exhibits the same normal coding potential as thymine [Warner et al, J. Bacteriol., Vol. 145, pp. 687-695 (1981); and Kunkel, PNAS, Vol. 81, pp. 1494-1498 (1984)]; (b) uracil can be removed to create an abasic site, and in single strand DNA, an abasic site is lethal, i.e., biologically inactivated [Schaaper et al, PNAS, Vol. 78, pp. 1773-1777 (1981); and the Kunkel paper cited above]; and (c) the development of a uracil-containing DNA template [Sagher et al, Biochemistry, Vol. 22, pp. 4518-4526 (1983) and Tye et al, PNAS, Vol. 75, pp. 233-237 (1978)].